Under Aim 1 of this project, the necessary preclinical work for the initiation of a clinical trial testing CD22-targeted CAR T cells as a therapy for relapsed or resistant pre-B ALL has been completed. CD22 is expressed on 95 % of pre-B cell ALL and there is extensive expertise in the Pediatric Oncology Branch in CD22-targeted therapy for ALL using immunotoxin conjugated antibodies. A number of important findings resulted from the pre-clinical work that have substantially streamlined the process of generating CAR T cells. This trial is currently open and utilizes lentiviral-based viral vectors for genetic modification of patient-derived expanded T cells to be reinfused after lymphodepleting chemotherapy. The initial experience from this trial was published in Fry et al. Nature Medicine 2018 and demonstrated 70% complete remission rate at an active dose and was found to be effective in CD19 negative/dim/positive ALL. This trial established that the success of CD19 CAR T cell therapy can be extended to other targets on ALL. Additionally, since CD19 negative relapse has been observed following CD19 CAR therapy, a second target will potentially increase the overall curative potential of CAR therapy for ALL. This trial is ongoing, has treated 45 patients and a subsequent manuscript describing the extended experience, toxicity profile and implications of cell manufacturing changes is in development. This trial is exploring new scientific aims and is anticipated to remain open to optimize this treatment strategy. To date, it is the only highly effective CD22 CAR trial in pediatrics at this point in time. Through this trial, we also identified antigen modulation as a mechanism of relapse. Based on preclinical work performed in the Fry lab, which identified an agent to upregulate CD22, a future trial evaluating strategies to increase CD22 expression is in the planning stages. Under Aim 2 of this project we have developed a number of novel CAR constructs including a TSLPR-targeted CAR for which we hold a patent. This work has been published in Blood and we are preparing for a clinical trial. In addition, we have generated a CD19/CD22 bispecific CAR with excellent in vivo activity. This construct can prevent the emergence of target-loss variants of ALL in an in vitro system. This CAR is now actively being tested in the clinic, with two patients enrolled to date. This identical construct is also in clinical trials at Stanford for the treatment of pediatric ALL and diffuse large B cell lymphoma. We performed necessary pre-clinical work for the incorporation of an alternative, semi-automated T cell expansion and transduction that will further streamline the production process, which is being used for CAR manufacturing in this trial. Second, we are developing novel acute myelogenous leukemia CAR constructs that may reduce the on-target off-tumor toxicity associated with myeloid antigens such as Flt3 and CD33. The CD33 CAR trial is under active development with plans for clinical translation in 2019.